Cellulosic biomass is a favorable feedstock for fuel ethanol production because it is both readily available and less expensive than either corn or sugarcane. Nevertheless, substantial problems must be overcome before a typical cellulosic feedstock can be utilized effectively and economically as a substrate for the fermentative production of ethanol. By way of example, cellulosic biomass feedstocks may include wood pulp or agricultural residues, such as corn stover, straw, grass, or weeds. A typical feedstock is comprised of approximately 35-45% cellulose, 30-40% hemicellulose, 15% lignin and 10% of other components. The cellulose fraction is composed of linear (and to a substantial extent, microcrystalline) polymers of the hexose sugar, glucose. Saccharification of cellulose releases sugars, which may be converted into ethanol or other products by fermentation. The hemicellulose fraction is comprised mostly of pentose sugars, including xylose and arabinose.
Alcohol products derived from cellulosic biomass are relatively expensive when compared to analogous fuels from other sources. A significant cost factor is the need to provide hydrolyzing enzymes, such as cellulases, that attack the cellulosic and/or hemicellulosic substrates to release sugars. These enzymes are produced by microorganisms, and may be purified from fermentation broth. The cost of cellulase is presently a significant component of the overall cost of biomass-derived ethanol. In the United States, ethanol production is heavily subsidized by tax incentives that encourage the use of ethanol in reformulated gasoline.
A variety of cellulases are known. Table 1 below lists various cellulases of the Cel7 family. Cel7 enzymes are the principal component in commercial cellulase formulations—typically accounting for most of the actual bond cleavage in the saccharification of cellulose. Cel7 cellobiohydrolases are members of the Class of beta proteins, the Superfamily of concanavalin A-like lectins/glucanases, and the Family of glycosyl hydrolase family 7 catalytic core proteins. The Cel7 family of enzymes may differ from one another by various insertions, deletions, and alterations in the catalytic domain and linker peptide. The cellulose binding domain of Cel7 enzymes is highly conserved. Cel7A from Trichoderma reesei is the most widely used CBH I commercial enzyme because it is capable of withstanding commercial process conditions and demonstrates the highest known level of saccharification in the entire family.
TABLE 1VARIOUS CELLULOSE 1,4-β-CELLOBIOSIDASE MEMBERSGenBank/GenPeptEnzymeOrganismAccessionsSwiss ProtCellobiohydrolase IAgaricus bisporusZ50094 CAA90422.1Q92400exoglucanase C1Alternaria alternataAF176571 AAF05699.1Cellobiohydrolase IAspergillus aculeatusAB002821 BAA25183.1O59843Cellobiohydrolase (CbhA)Aspergillus nidulansAF420019 AAM54069.1Cellobiohydrolase (CbhB)Aspergillus nidulansAF420020 AAM54070.1Cellobiohydrolase A (CbhA)Aspergillus nigerAF156268 AAF04491.1Cellobiohydrolase B (CbhB)Aspergillus nigerAF156269 AAF04492.1Q9UVS8Cellobiohydrolase IClaviceps purpureaY07550 CAA68840.1O00082Cellobiohydrolase ICochliobolus carbonumU25129 AAC49089.1Q00328Cellobiohydrolase ICryphonectria parasiticaL43048 AAB00479.1Q00548Cellobiohydrolase I (Cel7A)Fusarium oxysporumL29379 AAA65587.1P46238Cellobiohydrolase 1.2Humicola griseaU50594 AAD11942.1O94093AAN19007.1Cellobiohydrolase IHumicola griseaD63515 BAA09785.1P15828X17258 CAA35159.1Q12621Cellobiohydrolase IHumicola griseaAB003105 BAA74517.1O93780var. thermoideaCellobiohydrolase I.2Humicola griseaAF123441 AAD31545.1var. thermoideaCellobiohydrolase IMelanocarpus albomycesCellulose 1,4-β-Melanocarpus albomycesAJ515705 CAD56667.1cellobiosidase (Cel7B)Cellobiohydrolase INeurospora crassaX77778 CAA54815.1P38676CellobiohydrolasePenicillium funiculosumAJ312295 CAC85737.1Cellobiohydrolase IPenicillium janthinellumS56178 CAA41780.1Q06886X59054 CAA41780.1CellobiohydrolasePhanerochaete chrysosporiumS40817 AAA09708.1Q01762X54411 CAA38274.1Cellobiohydrolase I-1Phanerochaete chrysosporiumM22220 AAB46373.1P13860Z22528 CAA80253.1Cellobiohydrolase I-2 (Cel7D)Phanerochaete chrysosporiumL22656 AAA19802.1Q09431Z11726 CAA77789.1Z11733 CAA77795.1Z22527 CAA80252.1Z29653 CAA82761.1Z29653 CAA82762.1Cellobiohydrolase 1 (Cbh1A)Talaromyces emersoniiAF439935 AAL33603.2AY081766 AAL89553.1Cellobiohydrolase I (Cel7A)Trichoderma reeseiX69976 CAA49596.1P00725(Hypocrea jecorina)Cellobiohydrolase ITrichoderma virideX53931 CAA37878.1P19355Cellobiohydrolase ITrichoderma virideAB021656 BAA36215.1O93832Cellobiohydrolase I (CbhI)Volvariella volvacea V14AF156693 AAD41096.1
Cellulases often demonstrate enzymatic synergy in mixtures with other hydrolyzing enzymes; for example, between one enzyme that attacks cellulose and another that attacks hemicellulose. Various efforts have been made to provide transgenic organisms with one or more recombinant genes and obtain multiple functionality from a single organism, for example, as described in U.S. Pat. No. 5,536,655 issued to Thomas et al. for the gene encoding EI endoglucanase from Acidothermus cellulolyticus. 
United States patent application publication US 2002/0155536 to Van en Brink et al. discloses a method of isolating DNA sequences coding for one or more proteins of interest advantageously using an Aspergillus host. More specifically, cDNA is prepared from an organism of interest. Fragments of the cDNA are inserted into a vector to obtain a cDNA library. Subsequent transformation of the cDNA library into filamentous fungi, such as Aspergillus, facilitates screening for clones that express proteins of interest.
The '536 patent publication describes an expression system using filamentous fungi, such as Aspergillus, to provide host cells to screen for proteins of interest. Expression in an Aspergillus host renders the cloned polypeptide sequences more easily detectable due to a higher secretory capacity and less glycosylation, as by way of example, in Aspergillus nigeras compared to yeast. The '536 patent does not teach that P. funiculosum Cel7A can be secreted from Aspergillus awamori with full functionality.
United States patent application publication US 2002/0061560 to Lawlis describes a method of obtaining a secretory protein at a higher level in filamentous fungi, for example, Aspergillus awamori. More specifically, the coding sequence for the protein of interest is fused with DNA fragments encoding signal peptide, a cleavable linker peptide, and a portion of a protein native to the filamentous fungal host (i.e., protein that is normally secreted from Aspergillus). The '560 publication pertains to increased quantities of secreted proteins, and does not teach that P. funiculosum Cel7A can be secreted with full functionality.
WO 92/06209 to Ward et al. relates to an improved process for transforming the filamentous fungus T. reesei. T. reesei cells are treated with homologous DNA originally derived from T. reesei. The homologous DNA is provided with a selectable marker, which is used to select transformants. Although CBH I is used as an example, nothing related specifically to the processing and secretion of P. funiculosum Cel7A is taught or disclosed.
Efforts in recombinant technologies that pertain to the production of cellulases emphasize the production of cellulase in greater quantity or the production of cellulase having greater activity measured as conversion efficiency over time. It is notoriously difficult to compare the activity or performance of cellulases on naturally occurring cellulosic substrates. The naturally occurring substrates vary in composition, which makes it difficult to provide a uniform basis of comparison. Additionally, one is prone to draw unwarranted conclusions where higher concentrations of enzymes may produce surface effects when the enzyme interferes with itself. Similarly unwarranted conclusions may be drawn where adsorption effects (i.e., enzyme loss) impair the activity of lower cellulase concentrations. When it becomes necessary to measure performance with exactitude, commercial enterprises often choose to consult institutions, such as the National Renewable Energy Laboratory located in Golden, Colorado.
Transgenic expression of genes does not necessarily result in the production of useful cellulase. For example, glycosylation by yeast used to express the Cel7 family enzymes may render the enzymes less effective or ineffective. The choice of host organism is limited to those organisms that can survive commercial process conditions, for example, the Direct Microbial Conversion (DMC) process or the Simultaneous Saccharification and Fermentation (SSF) process. In the DMC method, a single microbial system both produces cellulase and ethanol as a fermentation product. The SSF method utilizes two biological elements, one that produces cellulase enzyme and the other, which ferments sugar to ethanol. The DMC process is described in (Brooks et. al., Proc. Annu. Fuels Biomass Symp., 2nd (1978). The SSF process is described in Ghose et. al., Biotechnol. Bioeng., (1984), 26 (4): 377-381(1984)., e.g., as described by Spindler et al, Biotechnology Letters, 14:403-407 (1992). By way of example, SSF process conditions may impose a pH of 4.5 to 5.5 and a temperature from 30° C. to 38° C. It can be difficult to choose a suitable host capable of both expressing a useful form of the cellulase of interest and surviving the process conditions where the cellulase is also active under process conditions.